Electrophoresis has proven to be an important and useful separation method for organic macromolecules such as proteins and nucleic acids. Separation occurs as the structure, size, shape or electrical charge of a molecule affects the relative mobility of the molecule through a viscous medium within an electrical field. The separation medium is typically a gel, but a viscous liquid can serve as a separation medium as well. Viscous liquids can be advantageous when separating very large macromolecules, viruses, or cells, or when rapid elution of the desired molecule from the separation medium is desired. However, heat produced during electrophoresis renders liquid media more susceptible than gels to distortion of the resultant separation pattern.
There are many ways to contain separation media for use in vertical electrophoresis systems, but all are limited to use with gelled separation media. Often, a casting device is used to prepare a gel-filled cassette. The gel-filled cassette is then removed from the casting device and placed in the vertical electrophoresis unit for use. In a typical casting device, the separation medium is prepared by pouring a gelling solution such as agarose or acrylamide into a cassette comprised of two plates offset by spacers, forming a mold with an open top and sealed bottom. When the gel-filled cassette is removed from the casting device and placed into the vertical electrophoresis unit, the top edge of the gel is typically exposed to an electrolyte solution in an upper buffer chamber of the unit and the lower edge of the gel is typically exposed to an electrolyte solution in a lower buffer chamber in the unit. When using agarose gel as a separation medium, the surface of the plates used in the cassette are usually treated or “frosted” to prevent the gel from slipping out of the cassette during electrophoresis. The spacers are typically flat narrow strips of a dielectric material that, when sandwiched between two plates with moderate force, form a liquid tight seal, thus serving as the sidewalls of the internal space within the cassette. Because side spacers do not interfere with the path of electric current across the separation medium, the side spacers remain in place during electrophoresis.
In order to contain the separation medium while it gels, physical blocking means are normally placed along the bottom of the cassette for filling. Because the bottom edge of the separation medium must be in electrical contact with an electrolyte-containing buffer solution when it is used in an electrophoresis unit, most physical blocking means are removed before the cassette is used in an electrophoresis unit. This requires additional manipulation of the cassette.
In many labs, empty cassettes are loaded manually. For example, a gasket or tape is used to seal the bottom opening of the empty cassette while the separation medium is poured into the cassette and allowed to gel. The tape or gasket is then removed prior to use in electrophoresis. Alternatively, some lab technicians use a bead of agarose the seal the bottom opening. Once the bead of agarose forms a solidified plug, the cassette is filled with separation medium. The cassette can then be used for electrophoresis as is. While manual methods are useful, they are cumbersome and require extensive manipulation. In addition, it is difficult to ensure the integrity of the bottom seal prior to filling the cassette when using manual techniques.
Manipulation of cassettes, whether prepared manually or using a casting device, prior to electrophoresis has several drawbacks. Manipulation complicates procedures in the laboratory thereby making tasks more difficult. Excessive or careless manipulation can also compromise the integrity of the cassette. For example, the risk of dropping or otherwise breaking a cassette increases with additional manipulation. When using gradient-type separation media, the integrity of the cassette is especially susceptible to manipulation even when cassettes are handled carefully. In addition, manipulation of cassettes increases the likelihood and amount of contamination from DNA and proteins that are carried on hands, gloves, and other surfaces. Another drawback of handling cassettes is that it increases the exposure of laboratory personnel to potentially hazardous chemicals such as acrylamide.
Another drawback with vertical electrophoresis methods is that most vertical systems are not well suited for large format gels. In other words, the cassettes for vertical systems are small compared to the separation area in a typical horizontal system. Many casting devices for vertical electrophoresis function by creating downward pressure on the glass plates to force the bottom of the cassette against a sealing gasket. Due to the amount of force required to achieve an effective seal, cassettes prepared using this type of casting device use relatively small glass plates in order to prevent cracking and breakage. Small cassettes are not acceptable for some specimens. For example, large separation areas are often required to adequately separate macromolecules in a complex mixture.
Some prior art systems cast the gel slab within the vertical electrophoresis unit. For example, in U.S. Pat. No. 5,709,788, Chen shows a system that casts a gel and conducts electrophoresis in a single enclosure. Chen uses air pressure to retain the gelling solution in the mold during polymerization of the gel. Then, the air pressure is released to allow an electrolyte solution to make contact with the gel. Chen's system is limited to electrophoresis applications using gelled media and cannot be used with viscous liquid media.
U.S. Pat. No. 5,882,495 issued to Garrels discloses another system that cast the gel and conducts electrophoresis in the same unit. Garrels teaches an electrophoresis platform that tilts between horizontal and vertical positions. An opening in the lower reservoir of the device is scaled with a reversible sealant, such as tape, during polymerization of the gelling solution. The reversible sealant (e.g., tape) is removed or cut prior to electrophoresis. The Garrels system is limited to use with gelled media.